Effect And Mechanism Of Triptolide On Renal Fibrosis After Acute Renal Ischemia-reperfusion Injury

Objective:Renal fibrosis(RF)is the common underlying cause of all progressive kidney diseases,and also the progression of Chronic kidney disease(CKD)to End stage kidney disease,Important pathological basis of ESKD.According to the theory of traditional Chinese medicine,RF treatment needs to promote blood circulation and remove blood stasis.Tripterygium wilfordii has the effect of dispelling wind and clearing arteries.It is used in the treatment of a variety of kidney diseases in clinic,and shows certain effect of inhibiting RF,which has a good prospect of development and application.However,the material basis and action mechanism of this medicine are still unclear.The purpose of this study was to analyze the material basis and mechanism of anti-renal fibrosis of Tripterygium wilfordii by network pharmacology.The pharmacodynamic material basis and potential mechanism of Tripterygium wilfordii in the treatment of RF were preliminarily verified by in vitro experiments.In vitro and in vivo experiments were conducted to verify the mechanism of triptolide improving RF by inhibiting Wnt/β-catenin signaling pathway.Methods:1.Network pharmacology study of Tripterygium wilfordii against renal fibrosisNetwork pharmacology was used to study the material basis and pathways of action of Tripterygium wilfordii in the treatment of RF.The TCMSP and TCMID databases were used to screen the components contained in Tripterygium wilfordii,and the target genes corresponding to the components contained in Tripterygium wilfordii were obtained according to the Uniprot database.The RF-related targets were collected using Gene Cards,Dis Ge NET and OMIM databases.The targets corresponding to the components contained in Tripterygium wilfordii and the RF-related targets were matched and mapped to obtain The targets of Tripterygium wilfordii for the treatment of RF.Used Cytoscape software to map the network of core components of Tripterygium wilfordii-intersecting targets and analyzed the topological features of the network by Network Analysis plug-in to screen the core components of Tripterygium wilfordii.Imported the intersecting targets into STRING database to obtain the target interaction information for the treatment of RF and imported into Cytoscape software to construct Protein interaction network(PPI).GO and KEGG enrichment analysis of the intersecting targets used Metascape database.2.Mechanism of Tripterygium wilfordii core components in improving RF in HKC cellsHuman renal tubular epithelial cells(HKC)were treated with different concentrations of kaempferol(0,5,10,15,20 μmol/L),β-sitosterol(0,5,10,15,20 μmol/L)and triptolide(0,5,10,15,20 nmol/L)for 24 h.The cell survival rate was detected by CCK-8 assay,and the drug concentration with the highest cell survival rate was used as the dose concentration for subsequent experiments.The regulatory effects of three core components on the Wnt/β-catenin signaling pathway related target PLD-1 in HKC cells were investigated by Western blot and q-PCR.3.The effect and mechanism of triptolide on renal fibrosis in vitro and in vivo modelsCellular and animal renal fibrosis models were established.Renal fibrosis model of HKC cells was established by TGF-β induction.At the cellular level,Western blot analysis and q-PCR were used to study the effects of triptolide on the expression of α-smooth muscle actin(α-SMA)and collagen I(COL-I)protein and m RNA,and to explore the anti-renal fibrosis effect and mechanism of triptolide.On the animal level,Renal ischemia reperfusion injury(RIRI)mouse model was established,and the experiment was divided into three groups: Sham group,model group and triptolide group(100 μg/kg intraperitoneal injection once a day)were sacrificed after 7,14 and21 days of continuous administration.Kidney tissue and peripheral blood serum of mice were collected,serum creatinine(SCr)and urea nitrogen(BUN)were determined,and pathological tissue staining methods such as HE staining and Masson’s trichrome staining were performed on mouse kidney tissue to observe the effect of triptolide on kidney injury and renal fibrosis of RIRI mice.In addition,the expressions of α-SMA and COL-I genes related to renal fibrosis were detected by q-PCR and Western blot.The expression of PLD-1 and β-catenin in Wnt/β-catenin signaling pathway was further detected by q-PCR.Results:1.Network pharmacology study of Tripterygium wilfordii against renal fibrosisNetwork pharmacology combined with literature reports to screen and obtain a total of 27 core components in Tripterygium wilfordii,corresponding to a total of 133 target genes,3002 RF target genes and 76 intersecting genes.The three core components of Tripterygium wilfordii against renal fibrosis were obtained after network topology parameter screening: kaempferol,β-sitosterol and triptolide.GO and KEGG enrichment analyses revealed that multiple signalling pathways were involved in the Wnt pathway and others in the anti-kidney fibrosis of Tripterygium wilfordii.2.Mechanism of Tripterygium wilfordii core components in improving RF in HKC cellsThe results of CCK-8 assay showed that compared with 0 μmol/L,the survival rate of HKC cells decreased when the concentration of kaempferol ≥15 μmol/L,β-sitosterol ≥15 μmol/L and triptolide ≥10 nmol/L(P<0.01).Therefore,the follow-up experiment was conducted with kaempferol 10 μmol/L,β-sitosterol 10 μmol/L and triptolide 5 nmol/L as the administration concentration.Western blot and q-PCR results showed that triptolide could significantly reduce the expression levels of PLD-1 protein and gene(P<0.01).3.The effect and mechanism of triptolide on renal fibrosis in vitro and in vivo modelsAt the cellular level,morphological results showed that fusiform transformation increased in TGF-β group compared with control group(P<0.01);Fusiform transformation was reduced in TGF-β+ triptolide group compared with TGF-β group(P<0.01).In addition,Western blot and q-PCR results showed that compared with the control group,the expressions of α-SMA,COL-I proteins and genes expression in TGF-β group were significantly up-regulated(P<0.01),the COL-I and α-SMA protein and gene expressions of triptolide group were not statistically significant(P>0.05);Compared with TGF-β group,α-SMA and COL-I protein and gene expressions in TGF-β+triptolide group were significantly down-regulated(P<0.01).It is suggested that triptolide has no effect on the expression of EMT-related proteins and genes in normal HKC cells,but shows a strong inhibitory effect on the expression of EMT-related proteins and genes in HKC cells with RF development tendency.At the animal level,BUN and Scr indexes in model group were significantly up-regulated compared with those in Sham group.Compared with model group,BUN and Scr indexes in triptolide group were significantly decreased.HE showed that compared with Sham group,the renal tubular dilatation and glomerular atrophy in the kidney tissue of model group increased significantly.Triptolide group significantly improved the damage of kidney tissue.Masson’s trichrome staining showed that more collagen deposition and blue fibers were significantly increased in the renal tissue of model group.Triptolide inhibited collagen deposition in renal interstitium and alleviated pathological changes of renal fibrosis in mice.Western blot and q-PCR results showed that compared with Sham group,the protein and m RNA expression levels of α-SMA and COL-I in model group were significantly up-regulated(P< 0.01),suggesting that the renal tissue of the model group had obvious fibrosis.The protein and m RNA expression levels of α-SMA and COL-I in triptolide group were significantly down-regulated(P< 0.01),and with the extension of triptolide intervention time,the degree of relieving RF increased.The results of q-PCR showed that compared with Sham group,the expression levels of PLD-1 and β-catenin m RNA in model group were significantly increased(P< 0.01);Compared with model group,the m RNA expression levels of PLD-1 and β-catenin in triptolide group were significantly decreased(P< 0.01).Triptolide can inhibit the Wnt/β-catenin signaling pathway in vivo and show a strong inhibitory effect on the expression of EMT-related proteins,which can achieve anti-RF and protect the kidney.Conclusion:The core components of Tripterygium wilfordii,such as kaempferol,β-sitosterol and triptolide,can regulate Wnt signaling pathway to prevent and treat the occurrence and development of RF.Triptolide,as the active component of Tripterygium wilfordii,can significantly inhibit renal injury and fibrosis in RIRI renal fibrosis model and TGF-β-induced renal fibrosis model in vitro and in vivo,and this protective effect is related to the reduction of PLD-1 expression in renal tissue.Inhibition of Wnt/β-catenin signaling pathway activation and delay the occurrence and development of renal EMT.This project is a study of the effect and mechanism of piperlongine regulating ICAT in the treatment of renal fibrosis by Health Commission of Shanxi Province(No.2023121).